Much information has been learned about the osteoblast through the use of several cloned osteosarcoma cell lines which have the osteoblast phenotype and have provided convenient models of osteoblast behavior and function. No such cell line has been available as a model for the osteoclast. In part, this may be because the mature osteoclast is a terminal cell which does not proliferate. I have cloned a novel non-tumor forming cell line (FM-2) with osteolytic properties from a human osteosarcoma. Phenotypic characterization of this cell line as expressed in their ultrastructure, enzyme contents, multinucleation, calcitonin receptors (see Appendix) suggests that it is either an osteoclast precursor or a unique bone resorbing cell. The formation of spleen colony forming units when infused into irradiated rats and the phenotypic modulations shown by this cell when grown on three-dimensional collagen lattices further suggest that it may be an osteoclast "stem cell". The overall objective of this research proposal is to determine if it is a cell in the osteoclast lineage, to further characterize the osteoclastic properties of the FM-2 cell line and to define the conditions which permit maximal expression of these osteoclast-like properties. The experimental protocol is designed to: 1) identify and optimize the culture and substrate conditions which are critical for differentiation of the FM-2 cell line. This will be achieved by culturing the cells on devitalized bone or reconstituted substrates which may resemble bone or in an isolated in vivo system, the atriocular space of nude mice. A morphologic and biochemical profile to monitor phenotypic changes in the line or new sublines will be developed, 2) determine the effects of hormones active in bone (e.g., parathyroid hormone, calcitonin, or 1,25(OH)2D3) and differentiation factors (e.g., colony stimulating factor, OAF, 5-azacytidine, retinoic acid) on the phenotypic expression of the cell line, 3) restore bone remodeling function to microphthalmic mice by FM-2 cell grafts, 4) isolate and partially purify the bone resorbing factor using hydroxyapatite chromatography. The partially purified factor will be used to generate monoclonal antibodies for subsequent use in affinity chromatography and studies of the biological properties of the factor. These studies should provide new information on the differentiation of bone resorbing stem cells in vitro. The establishment of differentiated bone resorbing cells may prove useful as modes for understanding the biology and biochemistry of osteoclasts and other bone resorbing cells. Such models could also be applied to investigations of the mechanisms of bone loss in both physiologic and pathologic states.